EP1032663A1 - Procede d'evaluation des effets toxiques et pathologiques de stimuli environnementaux sur la transcription des genes - Google Patents

Procede d'evaluation des effets toxiques et pathologiques de stimuli environnementaux sur la transcription des genes

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Publication number
EP1032663A1
EP1032663A1 EP98954606A EP98954606A EP1032663A1 EP 1032663 A1 EP1032663 A1 EP 1032663A1 EP 98954606 A EP98954606 A EP 98954606A EP 98954606 A EP98954606 A EP 98954606A EP 1032663 A1 EP1032663 A1 EP 1032663A1
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EP
European Patent Office
Prior art keywords
genes
gene
mammal
cells
grid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP98954606A
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German (de)
English (en)
Inventor
Timothy SmithKline Beecham Pharmaceut. BERTRAM
Michael J. SmithKline Beecham Pharmaceut. BROWNE
Peter SmithKline Beecham Pharmaceut. BUGELSKI
Paul SmithKline Beecham Pharmaceuticals ENGLAND
Ian SmithKline Beecham Pharmaceuticals MITCHELL
Andrew SmithKline Beecham Pharmaceuticals RUT
Gwyn SmithKline Beecham Pharmaceuticals MORGAN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SmithKline Beecham Ltd
SmithKline Beecham Corp
Original Assignee
SmithKline Beecham Ltd
SmithKline Beecham Corp
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Publication of EP1032663A1 publication Critical patent/EP1032663A1/fr
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • the present invention relates to the use of arrays or grids of mammalian gene sequence fragments from genomic (or cDNA) libraries for the screening of environmental factors, such as pharmaceutical compounds, physical factors, infectious agents, etc, for a toxic or pathologic effect upon gene transcription.
  • Mammalian cells frequently respond to exogenous stimuli of many types by altering the rate of transcription. For example, exposure of mammalian cells to environmental factors such as ultraviolet light, pharmaceutical compounds and many others can increase or decrease the quantity of messenger RNA produced by the cells. These changes in transcriptional regulation can result in toxic or pathological responses by the mammal. For example, where the external stimuli is prolonged exposure to UV rays, the toxic response of the mammal can be sunburn. Where the external stimuli is a compound known to be hepatotoxic, the response is liver damage. Where the external stimuli is a carcinogen, the toxic response is uncontrolled growth of cells.
  • the invention provides a method of assessing the genetic effect of a selected environmental factor on a mammalian subject, said method comprising the steps of:
  • each grid comprising a surface on which is immobilized at predefined regions on said surface a plurality of unique defined gene sequence fragments, said oligonucleotide sequences comprising genes or fragments of genes obtained from a healthy member of said mammalian species;
  • step (c) extracting and isolating mRNA from said exposed cells, tissue or organ of step (b); (d) extracting and isolating control mRNA from mammalian cells, tissue or organ not exposed to said factor;
  • the method of the invention thus employs the following steps.
  • a plurality of identical DNA grids is prepared.
  • a plurality of defined amplified gene sequences (or oligonucleotide sequences) is immobilized.
  • These gene sequences preferably are known or unknown genes, or fragments of genes, obtained from the cells (or a library of cells) of a healthy member of the mammalian species.
  • Messenger RNA is isolated and extracted from mammalian cells which are not exposed to a selected environmental stimulus, thus forming the "control" RNA.
  • test mRNA is extracted from mammalian cells which have been exposed for a sufficient time to affect gene transcription to the selected stimulus.
  • the control and test mRNA are randomly labeled, and each mRNA preparation is applied to an identical grid.
  • the respective hybridization patterns are compared to identify any change in the test pattern from the control pattern, indicative of an effect on transcriptional regulation of the mammalian cells exposed to the stimulus.
  • the determination of stimuli having a toxic or pathologic effect is useful, e.g., in the screening and development of new pharmaceutical agents and therapies.
  • the arrays or grids of mammalian gene sequence fragments from genomic (or cDNA) libraries used in the method of the invention may be high density DNA arrays or grids.
  • the method described above is performed for a "class" of stimuli, e.g., chemical or pharmaceutical compounds, which are to generate a common toxic or pathologic effect upon exposure to mammalian cells, e.g., hepatotoxicity.
  • the method generates a "fingerprint" hybridization pattern for e.g., hepatotoxic, stimuli.
  • test candidate drugs compositions may be screened for the likelihood of causing hepatotoxicity in mammalian cells by comparing the test hybridization pattern to the fingerprint at an early stage in drug development.
  • the methods of the present invention may be performed to identify those genes which are the most responsive to a particular toxic effect of an external stimuli.
  • the invention provides methods of identifying possible toxic or pathological effects of a variety of disparate physical stimuli, as well as chemical and pharmaceutical stimuli.
  • the present invention meets the needs of the art by providing a method of assessing the effect of any environmental factor or stimulus on gene expression in a mammalian subject by using DNA gridding techniques.
  • DNA gridding techniques employed as described below, permit the identification of genes which display a response to a test compound, permit the identification of a hybridization pattern characteristic of known physiologic effect in response to a test compound and permit the "fingerprinting" of certain selected toxic effects.
  • the fingerprints are useful in screening new compounds or drug candidates for potential toxicity and in screening for the effect on gene transcription of other environmental stimuli.
  • gene refers to the genomic nucleotide sequence from which a cDNA sequence is derived.
  • the term gene classically refers to the genomic sequence, which upon processing, can produce different RNAs.
  • genomic library is meant to include, but is not limited to, plasmid libraries, PCR products from genomic libraries, cDNA libraries and known sequences. Methods for the construction of such libraries are well known by those skilled in the art. A genomic library may be adjusted to minimize the number of complete genes present in a single genomic insert to approximately one gene. Techniques for this adjustment are well known to the skilled artisan.
  • isolated means altered “by the hand of man” from its natural state; i.e., that, if it occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living animal in its natural state is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated,” as the term is employed herein.
  • isolated means that it is separated from the chromosome and cell in which it naturally occurs.
  • Phathogenic effect or “pathologic effect”, as used herein, refers to a change in gene expression which may cause a disease or disorder. The change is due to exposure of a mammal or mammalian cell to some environmental stimulus, as detailed below.
  • solid support refers to any substrate which is useful for the immobilization of a plurality of defined materials derived from a genomic library by any available method to enable detectable hybridization of the immobilized polynucleotide sequences with other polynucleotides in the sample.
  • solid supports one desirable example is the support described in International Patent Application No. WO91/07087, published May 30, 1991.
  • other useful supports include, but are not limited to, nitrocellulose, nylon, glass, silica and Pall BIODYNE C membrane. It is also anticipated that improvements yet to be made to conventional solid supports may also be employed in this invention.
  • grid means any generally two-dimensional structure on a solid support to which the defined materials of a genomic library are attached or immobilized.
  • three types of grids are useful.
  • One grid useful in this invention contains as its defined oligonucleotide materials, unique nucleic acid sequences [or “tags”; or expressed sequence tags (“EST”)] from all human genes identified.
  • a second useful grid contains unique nucleic acid ESTs from genes cloned from a tissue or a cell line.
  • Still a third type of grid useful in the present invention contains unique nucleic acid tags from genes classified as particularly relevant to identification of a selected environmental toxicity. Grids are desirably constructed from animal species used in the preclinical assessment of compound safety.
  • predefined region refers to a localized area on a surface of a solid support on which is immobilized one or multiple copies of a particular amplified gene region or sequence and which enables hybridization of that clone at the position, if hybridization of that clone to a sample polynucleotide occurs.
  • immobilized it is meant to refer to the attachment of the genes to the solid support. Means of immobilization are known and conventional to those of skill in the art, and may depend on the type of support being used.
  • the terms "environmental factor” or “environmental stimuli” are used herein to describe a wide variety of physical, chemical or biological factors which cause changes in gene transcription in a mammalian cell when the mammal itself, or a culture of such mammalian cells, is exposed to that factor.
  • physical environmental stimuli can include, without limitation, the diet of the mammal, an increase or decrease in temperature; an increase or decrease in exposure to ionizing or ultraviolet radiation, and the like.
  • a biological/chemical stimuli can include, without limitation, administering a transgene to the mammal, or eliminating a gene from the mammal; administering an exogenous synthetic compound or exogenous agent or an endogenous compound, agent or analog thereof to the mammal.
  • an exogenous synthetic compound can be a pharmaceutical compound, a toxic compound, a protein, a peptide, a chemical composition, among other.
  • An exogenous agent can include natural pathogens, such as microbial agents, which can alter gene transcription.
  • pathogens include bacteria, viruses, and lower eukaryotic cells such as fungi, yeast, molds and simple multicellular organisms, which are capable of infecting a mammal and replicating its nucleic acid sequences in the cells or tissue of that mammal.
  • pathogens include bacteria, viruses, and lower eukaryotic cells such as fungi, yeast, molds and simple multicellular organisms, which are capable of infecting a mammal and replicating its nucleic acid sequences in the cells or tissue of that mammal.
  • pathogen is generally associated with a disease condition in the infected mammal.
  • An endogenous compound is a compound which occurs naturally in the body. Examples include hormones, enzymes, receptors, ligands, and the like.
  • An analogue is an endogenous compound which is preferably produced by recombinant techniques and which differs from said naturally occurring endogenous compound in some way.
  • transcriptional effect is meant an increase or decrease in rate of transcription in the mammalian cells exposed to the stimuli.
  • a “fingerprint” as used herein is defined as a characteristic hybridization pattern on a grid indicating a common toxicological response, i.e., similar increases in gene transcription that result in similar tissue damage. For example, using the methods described herein, one may generate a "hepatotoxic" fingerprint, which can be used to identify compounds which are likely to have a toxic effect on the liver, and so on.
  • label as used herein is meant any conventional molecule which can be readily attached to mRNA and which can produce a detectable signal, the intensity of which indicates the relative amount of hybridization of the mRNA to the DNA fragment (oligonucleotide) on the grid.
  • Preferred labels are fluorescent molecules or radioactive molecules. A variety of well-known labels can be used.
  • a method which enables the association of selected environmental stimuli with changes in gene transcription.
  • One of the specific applications of this technology is the understanding and prediction of toxic reactions to environmental manipulations and modifications, such as those stimuli listed above.
  • Another application is in pre-clinical and clinical drug development, where the method of this invention enables the screening of compounds having a similar toxic effect on gene transcription by comparison to the effect of another stimulus.
  • each grid carries on its solid surface a plurality of defined unique gene (oligonucleotide) sequences immobilized at predefined regions on the surface.
  • the gene sequences immobilized on the grids are as defined above, i.e., as unique nucleic acid tags from all human or other mammalian genes, or from only a selected tissue, e.g., reticulocytes, or the liver, or a selected cell line, or from genes known to be relevant to environmental toxicity, e.g., the lung, kidney, heart, blood cells, etc.
  • genes or fragments of genes immobilized on the grids may be obtained from an oligonucleotide library of a healthy member of the mammalian species, e.g., a healthy human.
  • mammals of interest include, without limitation, a non-human primate, a rodent, and a canine.
  • the grids reflect a single target organ, although such a specific target grid can be used. It is anticipated that the response of the mammalian cell to various environmental stimuli that effect gene transcription is likely to be stereotypic of genes in other cells.
  • the grid can be prepared from red or white blood cells, reticulocytes, or undifferentiated cells, even where the particular toxicological effect is damage to the liver or some other particular tissue.
  • such a grid can be prepared from hepatocytes only, or from cells from the effected organ or tissue only. All grids are anticipated to reflect the same hybridization pattern upon exposure to a reagent or stimulus that is known as hepatotoxic. The same is true regardless of the type of toxicological damage, e.g., cardiac damage, kidney damage, hematopoietic cell damage, etc.
  • the gene fragments immobilized on the grid may be obtained from a random cDNA library of the target mammal using known techniques.
  • a cDNA library of genes from a selected organ or tissue may be prepared as the source of the sequences immobilized on the grid.
  • the RNA is isolated and reverse transcribed to cDNA using standard procedures for molecular biology such as those disclosed by Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed; Cold Spring Harbor Laboratory Press, Cold Spring Harbor Lab Press, Cold Spring Harbor, NY 1989.
  • the cDNA library is then constructed in accordance with procedures described by Fleischmann et al. Science, 1995, 269:496- 512.
  • a cDNA library can comprise a plasmid library, PCR products from a cDNA library, or known sequences.
  • a plurality of genes or gene fragments, whether known or random and unknown, from the selected library are gridded onto a surface of a solid support at predefined locations or regions, preferably at 6X coverage.
  • plural of materials derived from the genomic library it is meant to include, but is not limited to, individual clones spotted onto and grown on a surface of the solid support at predefined locations or regions; or plasmid clones isolated from said library, PCR products derived from the plasmid clones, or oligonucleotides derived from sequencing of the plasmid clones, which are immobilized to the surface of the solid support at predefined locations or regions.
  • selection of genes involved in e.g., carcinogenicity, apoptosis, inflammation, metabolism of compounds etc may be used.
  • the grids used in the invention may contain, e.g., up to 5,000 genes or gene fragments.
  • the grids preferably contain up to 1,500 genes or gene fragments, e.g., 100 to 1,500 genes or gene fragments, more preferably about 1,000 genes or gene fragments.
  • binding substances Upon activation, these binding substances become capable of binding and immobilizing the materials derived from the genomic library.
  • Any of the known solid substrates suitable for binding nucleotide sequences at predefined regions on the surface thereof for hybridization and methods for attaching nucleotide sequences thereto may be employed by one of skill in the art according to the invention.
  • the genes or gene fragments may be of known or unknown function. In a fingerprinting method it is not necessary to know the function of every gene since the method may not be looking at specific pathways of toxicity but at distinct patterns of gene expression in response to environmental factors.
  • B. Obtaining the mRNA for hybridization to the grids
  • the selected mammalian cells, tissues or organs to be examined for transcription changes are subjected to the environmental stimulus for a sufficient time to affect transcription of messenger RNA in the cells.
  • This "exposing" step can occur by treating or exposing a living healthy animal or human to the stimulus.
  • the selected mammal may be administered a reagent, such as an exogenous or endogenous compounds as described above.
  • the mammal may be exposed to a physical stimulus, e.g., UV radiation.
  • a mammalian cell culture or tissue culture, or viable organ e.g., liver, heart, etc.
  • a control mRNA source is an untreated animal, tissue, organ or cell culture.
  • the exposure to the environmental stimulus which may be stimuli known to cause a specific physical effect, e.g., hepatocyte damage, cancer, etc., occurs for a time sufficient to result in the alteration from the normal of the transcription level of the cells so exposed.
  • the sufficient time will depend upon the particular stimulus being studied and, in fact, determination of a sufficient stimulus time is well within the skill of the art.
  • the culture is then incubated under a selected set of defined in vitro or in vivo conditions to produce a test culture.
  • non-exposed cells are also cultured under the same set of defined conditions to produce a control culture.
  • defined conditions it is meant, but is not limited to, standard in vitro culture conditions recognized as normal (i.e., non- pathogenic) for a selected mammalian cell, as well as in vitro conditions which reflect or mimic in vivo pathogenic settings (conditions) such as heat shock, auxotrophic, osmotic shock, antibiotic or drug selection/addition varied carbon sources, and aerobic or anaerobic conditions, and in vivo, pathogenic conditions.
  • such conditions are predetermined to allow maximum growth of the non- exposed cells.
  • the cells are then harvested from the animal, organ, tissue or cell culture by conventional means. Harvesting can be performed during various growth stages of the cells to ascertain the essentiality of a particular gene during different stages of growth. For example, harvesting can be performed during early logarithmic growth, late logarithmic growth, stationary phase growth or late stationary growth.
  • RNA or DNA
  • RNA is then extracted and isolated from the harvested non-exposed cells of the control culture, and RNA is extracted and isolated from the cells exposed to the stimulus of the test culture using standard methodologies well known to those skilled in the art.
  • mRNA extracted from the cells of the control culture and from the cells of the test culture are then used to generate labeled probes.
  • mRNA from the control and test cells When mRNA from the control and test cells is used to generate the probes, isolated mRNA is labeled according to standard methods using random primers, preferably hexamers, and reverse transcriptase. Such methods are routinely performed by those skilled in the art. All mRNA from the "control" or the "exposed” source is randomly labeled by conventional means, such as nick translation, multiprime labelling or other commonly used enzymatic labeling methodology. Known conventional methods for labelling the mRNA sequences may be used and make hybridization of the immobilized materials detectable. For example, fluorescence, radioactivity, photoactivation, biotinylation, energy transfer, solid state circuitry, and the like may be used in this invention. C. Hybridization to the grids
  • labeled mRNAs are then used as hybridization probes against the identical high density grids.
  • Labeled probes prepared from mRNA extracted from the test culture are hybridized to one grid to produce a "test" hybridization pattern.
  • Labeled probes from the mRNA extracted from the cells of the control culture are hybridized to a second identical grid, resulting in a "control" hybridization pattern.
  • the generated test hybridization patterns and control hybridization patterns are then compared.
  • the mRNA binds to certain genes or gene fragments in the grid in proportion to the expression of the mRNA of such genes in a normal cell.
  • the pattern is detectable by an increased quantity of detectable signal, e.g., fluorescence, at locations on the grid of those genes which are normally expressed in greater quantities that others in the remainder of the grid.
  • test compound or stimulus is a stimulus known to cause a physiological effect
  • a physiological effect for example, a toxic reaction of a subject resulting in damage to a major organ, e.g., liver, kidney, heart, blood cells
  • the method of this invention may be performed to provide a hybridization pattern which correlates with that damage.
  • any collection of known and structurally distinct toxicants which have the same physiological effects, e.g., hepatotoxicity can be employed in this method to generate a characteristic "fingerprint" hybridization pattern for hepatotoxic stimuli.
  • a set of grids are calibrated with a repertoire of the structurally diverse toxicants that produce the same pathological/toxicological reaction; e.g. hepatotoxicity or nephrotoxicity.
  • labeled RNA from a mammalian cell source exposed to the known toxicants are hybridized to identical grids to produce a common toxicant hybridization pattern.
  • the variety of known toxicants produce a characteristic common hybridization pattern, the common toxicological responses are likely to be the result of similar increases in transcription of selected genes, resulting in similar tissue damage.
  • This toxicological fmge ⁇ rint pattern may be used along with the "control" pattern for comparison with the pattern of a test compound/stimulus of unknown function or result.
  • the common fingerprint for, e.g., hepatotoxicity is used to screen a stimulus of unknown function or effect to determine if that stimulus is likely to produce hepatotoxicity in the mammal. Similarity in the "test" pattern to the hepatotoxic fingerprint enables the putative identification of the test compound as a hepatotoxic compound. Thus, if the test compound was a drug candidate, it can be eliminated from consideration at the earliest stages of drug development on the basis of its effects on gene transcription as measured on the grids. Similarly the method permits the test compound or stimulus, if an environmental factor present in e.g., the workplace, such as radiation, etc., to be identified as a potential health hazard, and corrected.
  • an environmental factor present in e.g., the workplace, such as radiation, etc. to be identified as a potential health hazard, and corrected.
  • a battery of finge ⁇ rint hybridization patterns may be prepared for all known toxicants. Any new drug candidate or other environmental stimulus may be screened by the above method for probable toxicological effects by comparison to standard finge ⁇ rints for other known stimuli causing liver damage, kidney damage, damage to the hematopoietic systems, etc. Such a screening method will enable quick and early evaluation of environmental stimuli, particularly new drug candidates.
  • Finge ⁇ rint hybridization patterns may be stored in a database and pattern matching performed by datamining.
  • in vitro effects of pharmacologically relevant concentrations of compounds on gene expression in blood cells are examined using the methods of this invention.
  • a gene expression finge ⁇ rint is developed through this methodology by exposing the nucleated blood cells, e.g., reticulocytes, white cells, to a variety of toxicants as described above. The resulting finge ⁇ rint is used subsequently to predict whether a novel compound is likely to also produce a similar pathological reaction. The information assists decisions about which compounds to take forward to clinical development, and enhances safety in the clinic through accurate and early prediction of toxicity.
  • An alternative embodiment of the method of this invention is to analyze the in vitro effects of pharmacologically relevant concentrations of compounds on gene expression in blood cells.
  • the method described above, and/or the finge ⁇ rints generated for certain selected toxicities may be useful in identifying novel genes that may have a significant impact on the compound's toxicity.
  • Application of the compositions and methods of this invention as above described also provides other compositions, such as any isolated gene sequence which is unusually reactive to the toxic result of one or more known toxicants.
  • the methods of this invention is useful in a clinical setting.
  • Gene expression grids may aid in the identification of the mechanism underlying the occurrence of pathological reactions and toxicity in a minority of patients during human trials.
  • gene expression in cells derived from patients/volunteers known to have experienced the adverse event in question during a clinical trial can be compared to gene expression from those who remained well.
  • mRNA is obtained from cells of the target organ, but may also include mRNA obtained from blood cells in which transcription can be altered, e.g., white blood cells.
  • An embodiment of the invention is any gene sequence identified by the methods described therein. These gene sequences associated with the toxic reaction are used to obtain full-length cDNA clones by conventional methods. The genes may be studied in greater detail; e.g. through sequencing and mutation analysis. These gene sequences may be employed in conventional methods to produce isolated proteins encoded thereby.
  • the DNA sequences of a desired gene invention or portions thereof identified by use of the methods of this invention are inserted into a suitable expression system.
  • a recombinant molecule or vector is constructed in which the polynucleotide sequence encoding the protein is operably linked to a heterologous expression control sequence permitting expression of the human protein.
  • appropriate expression vectors and host cell systems are known in the art for mammalian (including human), insect, yeast, fungal and bacterial expression. The transfection of these vectors into appropriate host cells, whether mammalian, bacterial, fungal or insect, or into appropriate viruses, results in expression of the selected proteins. Suitable host cells, cell lines for transfection and viruses, as well as methods for construction and transfection of such host cells and viruses are well-known. Suitable methods for transfection, culture, amplification, screening and product production and purification are also known in the art.
  • the essential genes and proteins encoded thereby which have been identified by this invention can be employed as diagnostic compositions useful in the diagnosis of a disease or infection by conventional diagnostic assays.
  • a diagnostic reagent can be developed which detectably targets a gene sequence or protein of this invention in a biological sample of an animal.
  • a reagent may be a complementary nucleotide sequence, an antibody (monoclonal, recombinant or polyclonal), or a chemically derived agonist or antagonist.
  • the essential genes of this invention and proteins encoded thereby, fragments of the same, or complementary sequences thereto may themselves be used as diagnostic reagents.
  • reagents may optionally be detectably labeled, for example, with a radioisotope or colorimetric enzyme.
  • Selection of an appropriate diagnostic assay format and detection system is within the skill of the art and may readily be chosen without requiring additional explanation by resort to the wealth of art in the diagnostic area.
  • genes and proteins identified according to this invention may be used therapeutically.
  • genes identified as essential in accordance with this method and proteins encoded thereby may serve as targets for the screening and development of natural or synthetic chemical compounds which have utility as
  • a compound capable of binding to a protein encoded by an essential gene thus preventing its biological activity may be useful as a drug component preventing diseases or disorders resulting from exposure of the mammalian cells to the environmental stimuli.
  • compounds which inhibit expression of an essential gene are also believed to be useful therapeutically.
  • compounds which enhance the expression of genes essential to the growth of an organism may also be used to promote the growth of a particular organism. Conventional assays and techniques may be used for screening and development of such drugs.
  • a method for identifying compounds which specifically bind to or inhibit proteins encoded by these gene sequences can include simply the steps of contacting a selected protein or gene product with a test compound to permit binding of the test compound to the protein; and determining the amount of test compound, if any, which is bound to the protein. Such a method may involve the incubation of the test compound and the protein immobilized on a solid support. Still other conventional methods of drug screening can involve employing a suitable computer program to determine compounds having similar or complementary structure to that of the gene product or portions thereof and screening those compounds for competitive binding to the protein. Identical compounds may be inco ⁇ orated into an appropriate therapeutic formulation, alone or in combination with other active ingredients.
  • compositions as well as suitable pharmaceutical carriers, and the like are well known to those of skill in the art. Accordingly, through use of such methods, the present invention is believed to provide compounds capable of interacting with these genes, or encoded proteins or fragments thereof, and either enhancing or decreasing the biological activity, as desired. Thus, these compounds are also encompassed by this invention.
  • IMAGE clones human derived cDNA sequences inserted into bacterial plasmids
  • the stocks were streaked out onto agar plates, and 3 colonies per clone were PCR screened with gene specific primers to determine which clones contained the correct sequences. Positive clones were then sequenced (ABI automated sequencer) and checked against the sequence database to ensure the clones were correct.
  • Six clones were prepared de novo by PCR from SB human cDNA. Rat, mouse and dog clones were prepared de novo by Reverse Transcriptase-PCR (RT-PCR) from species specific RNAs using gene specific primers and were also sequence confirmed. Stocks containing the correct clones were preserved as glycerol stocks.
  • the microarray comprises of: 77 sequences representing 45 different mammalian genes; and 5 yeast gene sequences.
  • 38 amplification cycles were carried out: 2 minutes @ 94 C initial soak (1 cycle); 35 seconds @ 94 C (autoincrement 1 sec per cycle); 30 seconds @ 55°C; 1 minute 45 seconds @ 72 C (autoincrement 1 sec per cycle) and a 10 minutes @ 72 C final extension period.
  • a 10 ul aliquot from each of the suspended PCR products was mixed with an equal volume of 11M NaSCN (J. T. Baker) and deposited into individual wells of 96-well microtiter plates (Nunc). Approximately 1 nl of each sample was arrayed in duplicate onto silanized (3-aminopropyl trimethoxy silane treated) glass slides using high-speed robotics (Molecular Dynamics Generation II Microarray System). The average diameter of each array element was measured at 215 microns with the spot- to-spot centers at a distance of 500 microns. After printing, the slides were allowed to air dry and then placed into a vacuum oven for 2 hours at 80°C. Prior to hybridization, the slides were washed for 10 minutes in isopropanol, boiled for 5 minutes in ddH2 ⁇ , and air dried.
  • Probes were prepared by simultaneous reverse transcription and labelling in the presence of a fluorophore. The reactions were carried out with a GibcoBRL Superscript II kit (Preamplification System for First Strand cDNA Synthesis) and the protocol was as follows:
  • a PCR reaction mix was prepared and kept on ice until required: 2ul XI 0 PCR buffer (supplied with kit), 2ul 25mM MgCl2, lul dNTP mix (to give 500uM final concentration of each of dATP, dGTP and dTTP, and a final concentration of 280uM of dCTP), 0.8ul Cy3TM dCTP (Amersham) to give a final concentration of 40uM and 2ul 0.1M DTT to give a total volume of 7.8ul.
  • RNA (1 1.2ul) was added, on ice, to the 7.8ul PCR reaction mix, mixed gently and then incubated at 39.5°C for 5 minutes.
  • 1 ul of Superscript IITM (200U/ul) was added, mixed gently, and the mix incubated at 39.5°C for a further 60 minutes.
  • a further lul of Superscript IITM was added and incubated at 39.5°C for another 60 minutes.
  • the reaction was terminated by heat inactivating the Superscript II at 68 C for 5 minutes.
  • RnaseH (2U/ul) was added and incubated at 39.5 C for 20 minutes and the probe cleaned up by running through a Quiaquick PCR column according to the manufacturers instructions.
  • Yeast control RNA's were made by in vitro transcription of cloned YGL097, YDR432, YML113, YFL021 and YGR014 cDNA's using a Riboprobe in vitro Transcription System (Promega). For quality assurance pu ⁇ oses, the yeast RNA's were added to the reaction at ratio's of 1 :100, 1 :1,000, 1 :5,000, 1 :10,000 and 1 :20,000 (wt/wt) respectively. After incubating the reaction at 39.5 C for 60 minutes, an additional lul of Superscript II RT was added and incubated at 39.5 C for a further 120 minutes.
  • the probe was dried down and resuspended in 12ul (for full-length cover slips) or 4ul (for small cover slips) of hybridisation buffer (5xSSC, 0.1% SDS, 0.25uM pN2 ⁇ ) and incubated at 100 C for 5 minutes.
  • the probe mixture was pipetted onto the microarray surface and covered with a glass cover slip and sealed with latex glue.
  • the microarray was transferred to a hybridisation oven and incubated at 42 C for 15 hours.
  • the glue and coverslip was removed whilst the microarray slide was immersed in a bath of low stringency buffer (2xSSC, 0.1% SDS) at room temperature and the slide incubated for 5 minutes. The slide was then washed in a high stringency wash (0.5xSSC, 0.1% SDS) on a flat bed shaker at room temperature for 5 minutes. After repeating the high stringency wash, the microarray slide was quicky placed in a 50ml Falcon tube and centrifuged (2 minutes at 200 x g) to remove any traces of wash buffer.
  • 2xSSC low stringency buffer
  • SDS low stringency buffer
  • Fluorescence from the microarray was detected and quantitated using a Molecular Dynamics Gen II scanner.
  • the fluorescent signal is measured as intesity per mm ⁇ .
  • a background measurement for each spot was taken in an area surrounding each spot.
  • the density for each spot was "normalised” by calculating the ratio of the spot density to the sum of all the spot densities and expressed as the nDxA (for normalised density per unit area).
  • the ratio (T/C) of the treated vs control values was calculated for each spot for each treatment and time point. This was done for spot set 1 and spot set 2 separately. Starting with spot set 1 sequences having T/C ratios of >2 and ⁇ 0.5 were identified as showing differential gene expression. If the signal was weak ( ⁇ 0.35) in both spot sets for both treated and control samples, that sample was removed from the analysis as being outside the detectable range.
  • the spot images of each of the identified sequences were examined for dust spots or other "noise" which would give an incorrect densitometric value. Each differentially expressed sequence was ranked according to fold increase/decrease.

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Abstract

L'invention porte sur un procédé d'évaluation des effets pathologiques de stimuli ou de réactifs environnementaux sur des cellules de mammifère par identification sur une grille d'ADN d'un motif d'hybridation en forme 'd'empreinte digitale'. Ledit motif est caractéristique de différents stimuli ou réactifs environnementaux chimiques ou structurels présentant un effet adverse commun sur la transcription des gènes. Différentes substances expérimentales sont testées pour rechercher un effet toxique similaire en comparant leur motif d'hybridation sur une grille similaire avec l'empreinte digitale.
EP98954606A 1997-11-20 1998-11-20 Procede d'evaluation des effets toxiques et pathologiques de stimuli environnementaux sur la transcription des genes Pending EP1032663A1 (fr)

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US6624197P 1997-11-20 1997-11-20
US66241P 1997-11-20
PCT/GB1998/003445 WO1999027090A1 (fr) 1997-11-20 1998-11-20 Procede d'evaluation des effets toxiques et pathologiques de stimuli environnementaux sur la transcription des genes

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WO (1) WO1999027090A1 (fr)

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KR100865664B1 (ko) * 2000-06-14 2008-10-29 비스타겐 인코포레이티드 간 줄기 세포를 사용하는 독성 타이핑
US7590493B2 (en) 2000-07-31 2009-09-15 Ocimum Biosolutions, Inc. Methods for determining hepatotoxins
JP2002065259A (ja) * 2000-08-24 2002-03-05 Shinya Watanabe 核酸標識方法および核酸標識用キット
FR2819524B1 (fr) * 2001-01-18 2003-07-04 Antonios Vekris Hybridation differentielle par competition
WO2002059560A2 (fr) * 2001-01-23 2002-08-01 Gene Logic, Inc. Methode et systeme de prediction de l'activite biologique, y compris de la toxicologie et de la toxicite de substances
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KR20020096559A (ko) * 2001-06-21 2002-12-31 이준호 알코올에 의해 특이적으로 그 전사발현이 유도되거나저해되는 알코올 민감성 유전자 발굴
AU2002365904A1 (en) * 2001-07-10 2003-09-04 Gene Logic, Inc. Cardiotoxin molecular toxicology modeling
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EP1476747A4 (fr) * 2002-01-31 2008-06-11 Ocimum Biosolutions Inc Modelisation d'hepatotoxicologie moleculaire
US7469185B2 (en) 2002-02-04 2008-12-23 Ocimum Biosolutions, Inc. Primary rat hepatocyte toxicity modeling
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AU1166199A (en) 1999-06-15
CA2310621A1 (fr) 1999-06-03
WO1999027090A1 (fr) 1999-06-03

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